Helix pomatia

Nineteen GSL standards, namely glucoiberin (GIB), glucolepidiin (GLP), progoitrin (PRO), epiprogoitrin (EPI), glucoraphanin (GRA), glucoraphenin (GRE), sinigrin (SIN), gluconapin (GNA), sinalbin (SNB), glucomoringin (GMR), glucobarbarin (GBA), glucotropaeolin (GTR), glucobrassicanapin (GBN), glucoerucin (GER), glucobrassicin (GBS), 4-hydroxyglucobrassicin (4HGBS), 4-methoxyglucobrassicin (4MGBS), neoglucobrassicin (NGBS) and gluconasturtiin (GNS), were purchased from Cfm Oskar Co. (Marktredwitz, Germany). Diethylaminoethyl (DEAE)- Sephadex-A25, sodium acetate, HCl, and aryl sulfatase (EC 3.1.6.1, type H-1) from Helix pomatia, were obtained from Sigma-Aldrich (St. Louis, MO, USA). HPLC grade acetonitrile, methanol, and water were obtained from Avantor Performance Materials (Center Valley, PA, USA).

The plasma samples were prepared according to Cuomo et al. [29 (link)]. A 0.2 mL aliquot of plasma was transferred to a clean microcentrifuge tube and spiked with 100 μL of a solution containing 1000 U of β-glucuronidase/sulfatase (EC 3.2.1.31) from Helix pomatia (Sigma, St. Louis, MO) in 0.1 M phosphate buffer (pH 6.86) and 50 μL of methanol to liberate free curcumin [30 (link)], as a substantial amount of curcumin is glucuronidated or sulfated [23 (link)]. For enzymatic hydrolysation of the phase-2 conjugates of curcuminoids, the resultant mixture was thoroughly vortexed and incubated at 37 °C for 1 h. In a subsequent incubation, curcuminoids were extracted with 1 mL of ethyl acetate, and the mixture was then vortexed for 1 min, followed by sonication in a water bath for 15 min. After 6 min of centrifugation at 15,000g, the resulting upper organic layer was transferred to a 2 mL microcentrifuge tube and evaporated at 30 °C under negative pressure in a centrifugal concentrator to remove residual solvent. This extraction procedure was repeated for a total of two extractions. After treating the dried extract with 100 μL of methanol, 10 μL were injected into the HPLC-MS/MS. “Salbutamol” (ISTD) was used as an internal standard to ensure data accuracy. All standard curcuminoids for the quantification were purchased from Sigma Aldrich, USA.

Fifty metabolites of PAH (mono-hydroxylated-PAH, OH-PAH), corresponding to the most common metabolized forms of the 16 PAH used for animal treatment, were assessed in urine samples using a previously published method^{25 (link)}. Briefly, urine samples were incubated overnight at 37 °C in present of sulfatase and glucuronidase from Helix pomatia (Sigma-Aldrich, Bormen, Belgium) at pH 5.6. Upon acidification with HCl 32%, the samples were further purified using successively a liquid-liquid extraction with ethyl acetate/cyclohexane (50:50, v/v) and a solid phase extraction onto an Envi-Chrom P cartridge (Sigma-Aldrich, Bormen, Belgium). OH-PAH were eluted with acetate/cyclohexane (50:50, v/v), evaporated, resuspended in cyclohexane/methanol/water (50:40:10, v/v/v). The methanol-water layer containing OH-PAH was evaporated until fully dried. The residue was then further derivatized with 20 µL MtBSTFA for 30 min at 60 °C and then 1 µL was injected into an Agilent 7890 A gas chromatograph equipped with an HP-5MS capillary column (30 m, 0.25 mm i.d., 0.25 µm film thickness), coupled with an Agilent 7000B triple quadrupole mass spectrometer operating in electron impact ionization mode^{26 (link)}.

Curcuminoids were extracted from plasma samples according to the method of Cuomo et al. [3 (link)] Briefly, the plasma samples were thawed for 30 min at room temperature. In the meantime, 50 µL of internal standard solution (250 ng/mL) composed of Curcumin-d6 (ISTD: CND Isotope, Pointe-Claire, QC, Canada) in methanol was added in an empty Eppendorf tube of 1.5 mL to which 0.2 mL of plasma and 100 µL of β-Glucuronidase/Arylsulfatase 10,000 U/mL from Helix pomatia (Sigma-Aldrich, Saint-Louis, MO, USA) diluted in phosphate buffer 0.1 M, pH = 6.86 were added. The tubes were vortexed for 1 min and incubated at 37 °C for 1 h to hydrolyze the phase-2 conjugates of curcuminoids [20 (link),21 (link)]. Then, curcuminoids were extracted two times from the mixture by adding 1 mL of ethyl acetate, then vortexed 1 min and sonicated for 15 min. After sonification, tubes were centrifugated at 15,000 g at room temperature for 6 min. The superior organic phase was then transferred, and the process was repeated a second time. The combined organic phase was evaporated under nitrogen stream. The curcuminoids extract was reconstituted in 100 µL of methanol, centrifuge at 5000 g for 5 min to remove any particles, and 50 µL of the supernatant was then transferred to HPLC vials.

Expansin was a gift from Professor Simon McQueen-Mason (University of York, York, UK); it was the α-expansin CsExp1 expressed in tomato plants and dissolved in pH 4.5 35 mM MES/35 mM Acetate buffer containing 200 mM NaCl [59 (link)].
Snail powder extract was prepared by mixing 50 mg of snail acetone power from the visceral hump of Helix pomatia (Sigma-Aldrich, Poole, Dorset, UK, Cat. No. S9764, now discontinued) into 1 mL of MES buffer 10 mM MES buffer containing 5 mM KCl and 1 mM CaCl₂ titrated to pH 5.0 with 1 M NaOH using a laboratory vortexer and incubating for 10 min at room temperature before centrifuging for 10 min at 7200× g and collecting the supernatant.